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Create IgG with Defined Glycoforms

Transglycosylation of IgG

TransGLYCIT enables specific IgG glycan remodeling and prepares antibodies with defined and homogenous glycoforms. The technology platform is based on transglycosylation using engineered endoglycosidase and glycosynthase enzymes for rapid and site-specific remodeling of IgG Fc-glycans. Using TransGLYCIT, IgG with defined glycoforms are obtained within three hours. With TransGLYCIT G2S2 Afucosylated containing the FucosEXO 16 enzyme, it is possible to generate afucosylated antibodies for direct comparison of antibodies with or without core fucose.

 

Transglycosylation Workflow

The IgG Transglycosylation Workflow

  1. Deglycosylation
    The Fc N-glycans are trimmed to the core GlcNAc using the IgG-specific Immobilized GlycINATOR® (EndoS2) enzyme that hydrolyses all Fc glycoforms, including high-mannose, hybrid, complex and bisecting glycans. To obtain afucosylated glycoforms, the optional Immobilized FucosEXO 16 enzyme hydrolyzes the α1-6 linked core fucose.
  2. Transglycosylation
    The engineered glycosynthase TransINATOR catalyzes the transglycosylation reaction between the oxazoline reactive G2S2 glycoform and the exposed core GlcNAc.

 

Transglycosylation of IgG with G2S2

 
Figure 1. Deconvoluted mass spectra of the Fc/2 fragment of native (top), deglycosylated (middle), and transglycosylated (bottom) trastuzumab display the N-glycan profile throughout the TransGLYCIT workflow. The mAb was digested with FabRICATOR and the subunits analysed with LC-MS using reversed phase chromatography (Agilent 1290; Waters BioResolve RP mAb 2.1 x 50 mm) and ESI-Q-TOF mass spectrometry (Bruker Impact II).

Glycoengineering of the IgG glycan profile is important for the development of next-generation therapeutic antibodies with enhanced or silenced Fc effector functions. The generation of homogenous preparations is however challenging and often requires a complex bioprocess development or time-consuming cell-engineering strategies.

 

Using the TransGLYCIT G2S2 kit, the glycan profile of a therapeutic antibody was remodeled within three hours and analyzed at subunit level using LC-MS (Fig. 1). The mass spectra show the heterogenous glycan profile of native trastuzumab (top) and the enzymatic glycan remodeling of the TransGLYCIT workflow. The mass shifts show complete deglycosylation to the core GlcNAc (middle) and transglycosylation of the antibody to generate homogenous G2S2 and G2S2F glycoforms using TransGLYCIT (bottom).

 

 

Afucosylated IgG Glycans using TransGLYCIT

 
Figure 2. Deconvoluted mass spectra of the Fc/2 fragment of native (top), deglycosylated (middle), and transglycosylated (bottom) trastuzumab display the N-glycan profile throughout the TransGLYCIT workflow. The mAb was digested with FabRICATOR and the subunits analysed with LC-MS using reversed phase chromatography (Agilent 1290; Waters BioResolve RP mAb 2.1 x 50 mm) and ESI-Q-TOF mass spectrometry (Bruker Impact II).

Structural studies have shown that the antibody glycan profile impacts the binding to Fcγ receptors and immunological responses including antibody dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Afucosylated antibodies lacking the core fucose show increased binding to activating FcγIIIa receptors and thus an elevated ADCC response, providing a potential higher clinical efficacy for indications where this activity is required.

 

To generate an antibody with afucosylated glycans, the TransGLYCIT G2S2 Afucosylated kit was used to remodel the glycan profile of trastuzumab and analyzed by LC-MS at subunit level (Fig. 2). The resulting mass spectra show the heterogenous glycan profile of native trastuzumab (top), initially carrying both afucosylated and fucosylated Fc glycans. The mass shifts indicate a complete deglycosylation and defucosylation to the core GlcNAc (middle). A homogenous and afucosylated G2S2 glycoform was generated using TransGLYCIT (bottom).

 

 

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Popular FAQ

Unfortunately no, the enzyme requires trimming of the Fc-glycan using GlycINATOR to enable access to the core fucose substrate.

Yes, the transglycosylation reaction can be performed on all subclasses of human IgG. The reaction is somewhat slower on IgG2 and longer incubation times may be necessary to obtain over 95% transglycosylation.

No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.

GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.

The TransGLYCIT platform is developed for transglycosylation of human IgG.

 

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TransGLYCIT Brochure

TransGLYCIT Brochure

TransGLYCIT is a platform for transglycosylation of native IgG.

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